Nucleic acid amplification with continuous flow emulsion

ABSTRACT

Embodiments of the present invention are directed to methods and devices/systems for amplifying genetic material and may include providing a water-in-oil emulsion in a continuous flow. The emulsion may include a plurality of water droplets comprising microreactors. Each of the plurality of microreactors may include a single bead capable of capturing a nucleic acid template, a single species nucleic acid template and sufficient reagents to amplify the copy number of the nucleic acid template. The method also includes flowing the emulsion across a first temperature zone and a second lower temperature zone to thermally process the microreactors to amplify the nucleic acid template by polymerase chain reaction.

CLAIM TO PRIORITY AND RELATED APPLICATIONS

This application is a divisional of U.S. patent application Ser. No.11/045,678, filed Jan. 28, 2005 (now U.S. Pat. No. 7,927,797). Thisapplication also claims the benefit under 35 U.S.C. §119(e) of U.S.provisional patent application No. 60/540,016, filed Jan. 28, 2004. Thisapplication is also related to the following application Nos.:60/443,471, filed Jan. 29, 2003; 60/465,071, filed Apr. 23, 2003;60/476,313, filed Jun. 6, 2003; 60/476,504, filed Jun. 6, 2003;60/476,592, filed on Jun. 6, 2003; 60/476,602, filed Jun. 6, 2003;60/497,985, filed Aug. 25, 2003. All patent and patent applications inthis paragraph are hereby fully incorporated by reference in theirentirety.

This application also incorporates by reference the following copendingU.S. patent application Nos. 10/767,899, filed Jan. 28, 2004;10/767,894, filed Jan. 28, 2004; and 10/767,779, filed Jan. 28, 2004.

FIELD OF THE INVENTION

Embodiments of the present invention relate to methods and systems forclonally amplifying nucleic acid templates from a single copy number toquantities amenable for sequencing, as well as methods and systems forcontinuous flow PCR using emulsion and solid support for immobilizingamplified nucleic acids.

BACKGROUND

The ability to amplify a plurality of nucleic acid sequences, such as agenomic library or a cDNA library, is critical given the current methodsof sequencing. Current sequencing technologies require millions ofcopies of nucleic acid per sequencing reaction, therefore, amplificationof the initial DNA is necessary before genomic sequencing. Furthermore,the sequencing of a human genome would require tens of millions ofdifferent sequencing reactions.

Current techniques and systems for in vitro genome amplification involvelaborious cloning and culturing protocols that have limited the utilityof genomic sequencing. Other techniques, such as PCR, while fast andreliable, are unable to amplify a mixture of different fragments of DNAa genome in a representative and clonal fashion.

While random primed PCR can be easily engineered to amplify a pluralityof nucleic acids in one reaction, this method is not preferred becausethe amplified product will be a mixture of different DNA fragments fromthe library. In addition, in a random PCR environment starting with aplurality of fragments, some DNA sequences are preferentially amplifiedat the expense of other sequences such that the amplified product doesnot represent the starting material. This problem with PCR may beovercome if each individual member of a library is amplified in aseparate reaction.

However, this approach may be impractical if many thousands of separatereaction tubes are required for the amplification process, as a genomiclibrary or cDNA library may include more than 1,000,000 fragments.Individual amplification of each fragment of these libraries inseparate, conventional reaction tube is not practical.

SUMMARY OF THE INVENTION

The present invention provides for methods and systems for amplifying aplurality of nucleic acids (e.g., each sequence of a DNA library,transcriptome, or genome) in a rapid and economical manner using, forexample, a means for encapsulating a plurality of DNA sampleseffectively individually in a microcapsule of an emulsion (i.e., a“microreactor”), performing amplification of the plurality ofencapsulated nucleic acid samples simultaneously, and releasing theamplified plurality of DNA from the microcapsules for subsequentreactions.

Preferably, in some embodiments of the invention, a plurality of suchmicroreactors include at least one capture bead (and preferably a singlebead). Each capture bead is preferably designed to have a plurality ofoligonucleotides that recognize (i.e., are complementary to) a portionof a nucleic acid template, and the amplification copies of thistemplate. It is preferred that any one capture bead contain only oneunique nucleic acid species.

Embodiments of the present invention provide methods and systems forperforming continuous flow amplification, specifically, encapsulatedcontinuous flow amplification. Such embodiments may be used with thermalor isothermal amplification reactions, for example, PCR, rolling circleamplification, whole genome amplification, nucleic acid sequence-basedamplification, and single strand displacement amplification. Inpreferred embodiments, the apparatus employs steady state heatgeneration and transfer elements and cross-flow emulsion dropletgeneration as described in detail herein.

Accordingly, in one embodiment of the invention, a method for amplifyinggenetic material includes providing a water-in-oil emulsion in acontinuous flow wherein the emulsion comprises a plurality ofwater-based droplets comprising microreactors. The plurality of themicroreactors may each include one or more species of nucleic acidtemplates, and sufficient reagents to amplify the copy number of one ofthe nucleic acid templates. The method may also include thermallyprocessing the emulsion by flowing it across stationary controlledtemperature zones to amplify nucleic acid templates by polymerase chainreaction.

In another embodiment of the present invention, an apparatus foramplifying genetic material includes at least one fluid delivery device,at least one first temperature zone to cycle a plurality ofmicroreactors each including one or more species nucleic acid templatesto a first temperature, at least one second temperature zone to cyclethe plurality of microreactors to second temperature lower than thefirst temperature, a first conduit for flowing at least a stream of oiltherein from a first reservoir and a second conduit for flowing at leasta water based PCR solution from a second reservoir out of an orifice andinto the first conduit creating a water-in-oil emulsion. The PCRsolution upon entering the first conduit comprises a plurality ofdroplets comprising the microreactors for performing polymerase chainreactions. A plurality of the microreactors each include one or morespecies of nucleic acid template.

In another embodiment of the invention, a cross-flow emulsificationapparatus includes a first inlet for receiving an oil flow from a firstconduit, an outlet for directing a water-in-oil emulsion out of theapparatus, a second inlet for receiving a water based PCR amplificationreaction mixture from a second conduit and an orifice for delivering PCRreaction mixture from the second conduit into the first conduit, to forma plurality of water-in-oil droplets comprising microreactors. Aplurality of the microreactors each include one or more nucleic acidtemplates and sufficient PCR amplification reaction mixture to produce aplurality of copies of nucleic acid template.

In another embodiment of the present invention, an apparatus foramplifying genetic material includes a water-in-oil emulsion in acontinuous flow wherein the emulsion comprises a plurality of waterdroplets comprising microreactors. A plurality of the microreactors mayinclude a single bead capable of capturing one or more nucleic acidtemplates, and sufficient reagents to amplify the copy number of the oneor more nucleic acid templates. The apparatus may also include thermalprocessing means for thermally processing the emulsion to amplifynucleic acid templates by polymerase chain reaction.

In another embodiment of the present invention an emulsion generatorincluding an emulsion oil supply, at least one syringe including a bodyand a plunger for dispensing a mixture for emulsifying into the emulsionoil, a cross-flow emulsification device for emulsifying the mixture, thedevice including an input attached to the output of the syringe, asyringe pump including an actuator capable of oscillating the plunger ofthe at least one syringe micrometer distances at a predeterminedfrequency along a length of travel of the plunger within the syringebody of the at least one syringe.

In another embodiment of the present invention, a method forsubstantially reducing clogging of a nozzle in syringe pump includesproviding a syringe pump having at least one syringe including a body, aplunger having a plunger axis and an exit nozzle, the body fordispensing a mixture of micron or less sized particles suspended in amedium, and oscillating the plunger of the syringe along the axis of theplunger for micrometer distances at a predetermined frequency along alength of travel of the plunger within the syringe body.

In another embodiment of the present invention, an emulsion generatorincludes an emulsion oil supply, at least one syringe including a bodyand a plunger for dispensing a mixture for emulsifying into the emulsionoil, a magnetically-attractive mixing element disposed in the body ofthe syringe, a cross-flow emulsification device for emulsifying themixture, the device including an input attached to the output of thesyringe and a device capable of moving an external magnetic forceaxially along body of the syringe while in close proximity to thesyringe body.

In another embodiment of the present invention an emulsion generatorincludes an emulsion oil supply, at least one syringe including a bodyand a plunger for dispensing a mixture for emulsifying into the emulsionoil, a magnetically-attractive mixing element disposed in the body ofthe syringe, a cross-flow emulsification device for emulsifying themixture, the device including an input attached to the output of thesyringe and a rotating drum having a magnet helically wound along thesurface of the drum. The surface of the drum is positioned adjacent thebody of the syringe.

In another embodiment of the present invention, a syringe pump includesan area for receiving at least one syringe, where the syringe includes abody and a plunger having a plunger axis. The syringe may be used fordispensing a mixture for emulsification into an emulsion oil. Thesyringe pump may also include an actuator capable of oscillating theplunger of the at least one syringe along the plunger axis micrometerdistances at a predetermined frequency along a length of travel of theplunger within body of the at least one syringe.

In another embodiment of the present invention, a syringe pump includesan area for receiving at least one syringe, where the syringe includes abody and a plunger having a plunger axis. The syringe may be used fordispensing a mixture for emulsification into emulsion oil. The syringepump may also include a magnetically attractive mixing element disposedin the body of the syringe and a rotating drum having a magnet helicallywound along the surface of the drum, wherein the surface of the drum ispositioned adjacent the body of the syringe.

Other objects, advantages, features and benefits of the invention willbecome more readily apparent by reference to the attached drawings andfollowing detailed description of the invention.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a system block diagram of a system for a PCR amplificationsystem according to one of the embodiments of the present invention.

FIG. 2 is a schematic of a linear emulsification system according to anembodiment of the present invention for creating an emulsion flow.

FIG. 3 illustrates a depiction of beads (see arrows) suspended inindividual microreactors according to some embodiments of the invention.

FIG. 4 is a schematic of a linear emulsifier apparatus according to anembodiment of the present invention.

FIG. 5A is front view of a mixing Tee for a linear emulsificationapparatus according to an embodiment of the present invention.

FIG. 5B is a side view of the mixing Tee of FIG. 5A.

FIG. 5C is a cross-sectional view of the mixing Tee of FIG. 5A.

FIG. 5D is an enlarged cross-section of the detail of the nozzle area ofthe mixing Tee of FIG. 5A.

FIG. 5E is a close-up, perspective view of mixing area of the mixing Teeof FIG. 5A.

FIG. 5F is a close-up, perspective view of an alternative design of themixing area of the Tee of FIG. 5A.

FIG. 6A is a first, side-perspective view of a syringe pump assemblyaccording to one embodiment of the present invention.

FIG. 6B is bottom-perspective view of the syringe pump assembly of FIG.6A.

FIG. 6C is a backside-perspective view of the syringe pump assembly ofFIG. 6A.

FIG. 6D is a second, side-perspective view of the syringe pump assemblyof FIG. 6A.

FIG. 6E is third, side-perspective view of the syringe pump assembly ofFIG. 6A.

FIG. 7A is a schematic of a circular thermal processing system.

FIG. 7B is a perspective view of the circular thermal processing systemof FIG. 8A.

FIG. 8A is a schematic of another example of a continuous flow thermalprocessing/cycling system according to another embodiment of theinvention.

FIG. 8B is an exploded-perspective view of the thermal processing systemof FIG. 7A.

FIG. 8C is an assembled, perspective view of the thermal processingsystem of FIG. 7B.

DETAILED DESCRIPTION OF INVENTION Brief Overview of Bead EmulsionAmplification

Bead emulsion amplification may be performed by attaching a template(e.g., DNA template) to be amplified, to a solid support, preferably inthe form of a generally spherical bead. The bead is linked to a largenumber of a single primer species that is complementary to a region ofthe template DNA and the amplification copies of this template.Alternately, the bead is linked to chemical groups (e.g., biotin) thatcan bind to chemical groups (e.g., streptavidin) included on thetemplate DNA and amplification copies of this template. SeeWO2004069849, herein incorporated by reference.

The beads are suspended in aqueous reaction mixture and thenencapsulated in a water-in-oil emulsion. The template DNA may be boundto the bead prior to emulsification, or the template DNA is included insolution in the amplification reaction mixture.

The emulsion may be composed of discrete aqueous phase microdroplets(i.e., microreactors, see above), e.g., averaging approximately 60 to200 μm in diameter, enclosed by a thermostable oil phase. Eachmicroreactor contains, preferably, sufficient amplification reactionsolution (i.e., the reagents necessary for nucleic acid amplification).An example of an amplification reaction solution would be a PCR reactionmixture (polymerase, salts, dNTPs; and may also preferably include apair of PCR primers (primer A and primer B). In some cases, the templateDNA is included in the reaction mixture. A subset of the microreactorpopulation preferably includes microreactors having a single DNA beadpreferably with an attached nucleic acid template. This subset ofmicroreactors is the basis for the amplification in some of thepreferred embodiments of the present application. In one embodiment, theremaining microreactors which do not contain template DNA will notparticipate in amplification.

PCR amplification and PCR primers may be present in an asymmetric ratiosuch as 8:1 or 16:1 (i.e., 8 or 16 of one primer to 1 of the secondprimer) to perform asymmetric PCR. The primer species that may be usedin the lower concentration level is the same primer species that may beimmobilized on the bead. This will increase the probability that anamplified copy of the template DNA will anneal to the bead. The ratio ofPCR primers may also be substantially equal for normal PCR. Theamplification reaction, such as PCR, may be performed using any suitablemethod.

After PCR, the beads containing the immobilized amplified DNA may berecovered. The emulsion may be broken to recover the beads. Theimmobilized product may be rendered single stranded by denaturing (byheat, pH etc.) which removes the complimentary A strand. The A primersare annealed to the A′ region of immobilized strand, and the beadscontaining the immobilized strands are loaded with sequencing enzymes,and any necessary accessory proteins. The beads are then sequenced usingrecognized pyrophosphate techniques (described, e.g., in U.S. Pat. Nos.6,274,320, 6,258,568 and 6,210,891, incorporated in toto herein byreference).

Template Design

In a preferred embodiment, the nucleic acid template to be amplified bybead emulsion amplification is a population of DNA such as, for example,a genomic DNA library or a cDNA library. It is preferred that eachmember of the DNA population have a common nucleic acid sequence at thefirst end and a common nucleic acid sequence at a second end. This canbe accomplished, for example, by ligating a first adaptor DNA sequenceto one end and a second adaptor DNA sequence to a second end of eachmember of the DNA population. The nucleic acid template may be of anysize amenable to in vitro amplification (including the preferredamplification techniques of PCR and asymmetric PCR). In a preferredembodiment, the template is about 150 to 750 bp in size, such as, forexample about 250 bp in size.

Binding Nucleic Acid Template to Capture Beads

A single stranded nucleic acid template to be amplified may be attachedto a capture bead. The amplification copies of the nucleic acid templatemay also be attached to a capture bead. As non-limiting examples, theseattachments may be mediated by chemical groups or oligonucleotides thatare bound to the surface of the bead. The nucleic acid (e.g., thenucleic acid template, amplification copies, or oligonucleotides) may beattached to the solid support (e.g., a capture bead) in any manner knownin the art.

According to the present invention, covalent chemical attachment of anucleic acid to the bead can be accomplished by using standard couplingagents. For example, water-soluble carbodiimide can be used to link the5′-phosphate of a DNA sequence to amine-coated capture beads through aphosphoamidate bond. Alternatively, specific oligonucleotides can becoupled to the bead using similar chemistry, and then DNA ligase can beused to ligate the DNA template to the oligonucleotide on the bead.Other linkage chemistries to join the oligonucleotide to the beadsinclude the use of N-hydroxysuccinamide (NHS) and its derivatives, forexample.

In an exemplary method, one end of a linker may contain a reactive group(such as an amide group) which forms a covalent bond with the solidsupport, while the other end of the linker contains a second reactivegroup that can bond with the oligonucleotide to be immobilized. In apreferred embodiment, the oligonucleotide is bound to the DNA capturebead by covalent linkage. However, non-covalent linkages, such aschelation or antigen-antibody complexes, may also be used to join theoligonucleotide to the bead.

As non-limiting examples, oligonucleotides can be employed whichspecifically hybridize to unique sequences at the end of the DNAfragment, such as the overlapping end from a restriction enzyme site orthe “sticky ends” of cloning vectors, but blunt-end linkers can also beused. These methods are described in detail in U.S. Pat. No. 5,674,743.It is preferred that the beads will continue to bind the immobilizedoligonucleotide throughout the steps in the methods of the invention.

Each capture bead is preferably designed to have a plurality ofoligonucleotides that recognize (i.e., are complementary to) a portionof the nucleic template, and the amplification copies of this template.It is preferred that any one capture bead contain only one uniquenucleic acid species.

The beads used herein may be of any convenient size and fabricated fromany number of known materials. Example of such materials include:inorganics, natural polymers, and synthetic polymers. Specific examplesof these materials include: cellulose, cellulose derivatives, acrylicresins, glass, silica gels, polystyrene, gelatin, polyvinyl pyrrolidone,co-polymers of vinyl and acrylamide, polystyrene cross-linked withdivinylbenzene or the like (as described, e.g., in Merrifield,Biochemistry 1964, 3, 1385-1390), polyacrylamides, latex gels,polystyrene, dextran, rubber, silicon, plastics, nitrocellulose, naturalsponges, silica gels, control pore glass, metals, cross-linked dextrans(e.g., Sephadex™) agarose gel (Sepharose™), and other solid phasesupports known to those of skill in the art. In preferred embodiments,the capture beads are beads approximately 2 to 100 μm in diameter, or 10to 80 μm in diameter, most preferably 20 to 40 μm in diameter. In apreferred embodiment, the capture beads are Sepharose beads.

Emulsification

Capture beads with or without attached nucleic acid template may besuspended in a heat stable water-in-oil emulsion. Furthermore, the sizeof the microreactors may be adjusted by varying the flow rate and speedof the components. Additionally droplet size can also be varied bychanging the viscosity of the emulsion oil, and also by the usingdifferent orifice sizes in the cross-flow emulsion generating part.

Various emulsions that are suitable for biologic reactions are referredto in Griffiths and Tawfik, EMBO, 22, pp. 24-35 (2003); Ghadessy et al.,Proc. Natl. Acad. Sci. USA 98, pp. 4552-4557 (2001); U.S. Pat. No.6,489,103 and WO 02/22869, each fully incorporated herein by reference.It is noted that Griffiths et al., (U.S. Pat. No. 6,489,103 and WO99/02671) refers to a method for in vitro sorting of one or more geneticelements encoding a gene products having a desired activity. This methodinvolves compartmentalizing a gene, expressing the gene, and sorting thecompartmentalized gene based on the expressed product. In contrast tothe present invention, the microencapsulated sorting method of Griffithis not suitable for parallel analysis of multiplemicrocapsules/microreactors because their nucleic acid product is notanchored and cannot be anchored. Since the nucleic acids of Griffithsare not anchored, they would be mixed together during demulsification.

The emulsion is preferably generated by adding beads to an amplificationsolution. As used herein, the term “amplification solution” means thesufficient mixture of reagents that is necessary to performamplification of template DNA. One example of an amplification solution,a PCR amplification solution, is provided in the examples below. It willbe appreciated that various modifications may be made to theamplification solution based on the type of amplification beingperformed and whether the template DNA is attached to the beads orprovided in solution. The oil used may be supplemented with one or morebiocompatible emulsion stabilizers including Agrimer AL22 and otherrecognized and commercially available suitable stabilizers.

In preferred aspects, the emulsion is heat stable to allow thermalprocessing/cycling, e.g., to at least 94° C., at least 95° C., or atleast 96° C. Preferably, the droplets formed range in size from about 5microns to 500 microns, more preferably, from about 50 to 300 microns,and most preferably, from about 100 to 150 microns. Advantageously,cross-flow emulsion generation allows for control of the dropletformation, and uniformity of droplet size.

The microreactors should be sufficiently large to encompass sufficientamplification reagents for the degree of amplification required.However, the microreactors should be sufficiently small so that asufficient number of microreactors, up to about 20,000,000 or more, eachcontaining effectively a single member of a DNA library, can be suppliedfrom a small number of conventionally available syringes that can fittogether on a syringe pump. Notably, the use of microreactors allowsamplification of complex mixtures of templates (e.g., genomic DNAsamples or whole cell RNA) without intermixing of sequences, ordomination by one or more templates (e.g., PCR selection bias; see,Wagner et al., 1994, Suzuki and Giovannoni, 1996; Chandler et al., 1997,Polz and Cavanaugh, 1998).

With the limitations described above, the optimal size of a microreactormay be on average 100 to 200 microns in diameter. Microreactors of thissize would allow amplification of a DNA library comprising about18,000,000 members supplied to the emulsion generator in a volume of 9mls contained in three 3 ml syringes.

Amplification

After encapsulation of the bead and PCR solution and template DNA in themicroreactor, the template nucleic acid may be amplified, while attached(preferably) or unattached to beads, by any suitable method ofamplification including transcription-based amplification systems (KwohD. et al., Proc. Natl. Acad Sci. (U.S.A.) 86:1173 (1989); Gingeras T. R.et al., WO 88/10315; Davey, C. et al., EP Publication No. 329,822;Miller, H. I. et al., WO 89/06700), “RACE” (Frohman, M. A., In: PCRProtocols: A Guide to Methods and Applications, Academic Press, NY(1990)) and one-sided PCR (Ohara, O. et al., Proc. Natl. Acad. Sci.(U.S.A.) 86.5673-5677 (1989)). Still other methods such asdi-oligonucleotide amplification, isothermal amplification (Walker, G.T. et al., Proc. Natl. Acad. Sci. (U.S.A.) 89:392-396 (1992)), NucleicAcid Sequence Based Amplification (NASBA; see, e.g., Deiman B et al.,2002, Mol Biotechnol. 20(2):163-79), whole-genome amplification (see,e.g., Hawkins T L et al., 2002, Curr Opin Biotechnol. 13(1):65-7),strand-displacement amplification (see, e.g., Andras S C, 2001, MolBiotechnol. 19(1):29-44), rolling circle amplification (reviewed in U.S.Pat. No. 5,714,320), and other well known techniques may be used in thepresent invention.

In a preferred embodiment, DNA amplification is performed by PCR. PCRaccording to the present invention may be performed by encapsulating thetarget nucleic acid with a PCR solution comprising all the necessaryreagents for PCR. Then, PCR may be accomplished by exposing the emulsionto any suitable thermal processing regimen known in the art. In apreferred embodiment, 30 to 60 cycles, and preferably about 60 cycles,of amplification are performed. It may be desirable, but not necessary,that following the amplification procedure, there may be one or morehybridization and extension cycles, which comprise a similar meltingtime but a longer extension time, following the cycles of amplification.Routinely, the template DNA is amplified until typically at least twomillion to fifty million copies, preferably about ten million to thirtymillion copies of the template DNA are immobilized on each bead.

In some embodiments, the method of the invention employs continuous flowPCR to amplify the nucleic acid template. Various methods of continuousflow PCR have been reported in, e.g., Park et al., 2003, Anal. Chem.75:6029-6033; Curcio and Roeraade, 2003, Anal. Chem. 75:1-7; Chiou etal., 2001, Anal. Chem. 73:2018-2021; U.S. Pat. No. 6,207,031; U.S. App.Pub. 2001/0020588; Lagally et al., 2001, Anal. Chem. 73:565-570; U.S.Pat. Nos. 6,361,671, 6,284,525, 6,132,580, 6,261,431, 6,045,676,6,143,152, 5,939,312; U.S. App. Pub. 2002/0068357; Schneegas et al.,2001, Lab on a Chip 1:42-49; Kopp et al., 1998, Science 280:1046-1049;Nakano et al., 1994, Biosci. Biotech. Biochem. 58:349-352; and Larzul inU.S. Pat. No. 5,176,203, all of which are incorporated herein byreference. Advantageously, continuous flow PCR greatly reduces samplehandling and reaction times, while it increases amplificationspecificity. However, previous flow systems utilized serial slugs, i.e.,slugs of reagent that completely fill the diameter of the flow tube,that are separated by similar full slugs of air and oil. In contrast,embodiments of the present invention are directed to a water-in-oilemulsion used in conjunction with a continuous flow PCR. Thewater-in-oil emulsion comprises microreactors, allowing clonalamplification of a large population of nucleic acids. The microreactorsare about 10 to 50 times smaller than the diameter of the flow tube sothat a very large number of them are present in the flow stream. Forexample, a 2 mm diameter flow tube can carry 2,000 microreactors per cmof length.

The continuous flow PCR methods of the invention can be used to amplifythe sequences of an entire genome or transcriptome on a singleinstrument in less than half the time required for traditional thermalprocessing. Continuous flow of the emulsion across a solid state heattransfer element permits efficient and rapid (e.g., 60 second) reactioncycle. A 60 cycle amplification for example, would take 1 hour. Invarious embodiments, the nucleic acid template can be diluted to obtaineffectively one copy of delivered template per microreactor, and a finalyield of 1,000,000 to 10,000,000 template copies per bead. As examples,the continuous flow methods of the invention can be used with thermalamplification reactions (e.g., PCR) or isothermal reactions (e.g.,rolling circle amplification, whole genome amplification, NASBA, stranddisplacement amplification, and the like).

Amplification Systems

In one aspect, the method of the invention is performed using a systemfor continuous flow amplification, e.g., continuous flow PCRamplification. This system includes a means for forming an emulsion ofan amplification reaction mixture in a stabilized biocompatible oil.

FIG. 1 illustrates a general block diagram of an emulsification system200, as well as a thermal processor 112 and bead filtering device 114,according to one embodiment of the present invention. As shown, anemulsion oil 102 is pumped via pump 104 into a cross-flow emulsifier106. The emulsifier emulsifies a PCR reaction mixture (having aplurality of beads) 108, which is supplied to the emulsifier via a pump110, creating a plurality of microreactors in the emulsion oil flow.Each microreactor preferably includes on average a single bead and aneffective single species nucleic acid template.

The plurality of microreactors may then be thermally processed via athermal processor 112 to amplify the DNA template. After amplification,the beads (each containing the amplified nucleic acid), may be filteredout of the emulsion flow via a bead filter 314, and thereafter processedfor subsequent sequencing.

FIG. 2 illustrates a schematic diagram of an exemplary emulsificationsystem 200. As shown, an emulsion oil 202 from an emulsion oil supply204, via filter 206, is pumped via pump 208 to a cross-flow emulsifier210. The system may be controlled by a microprocessor based controller(not shown), which may be a personal computer (PC), or other controller(e.g., analog) controller device. Accordingly, the controller maymonitor the pressure of the emulsion oil flow via pressure sensor 212,so that the flow rate of the oil may be regulated, and the generalstatus of the system determined (e.g., pump failures, leaks). The pumpis preferably precisely controlled (e.g., electronically) to maintain anexact and consistent speed (e.g., from 1-10 mls/min, and preferablyabout 3 mls/min). A pressure dampening tube 214 may be used to attenuatepressure fluctuations in the oil caused by the pump, prior to the firstcross-flow emulsifier.

The emulsion oil is supplied to the cross-flow emulsifier 210 (see also106, FIG. 1). In this particular embodiment, the emulsion oil is flowedthrough multiple (in this case, three) injection/mixing tees 216(although a single injection tee or any other number may also be used).Each tee receives a PCR/bead mixture from a corresponding syringe 218. Asyringe pump 220 may be used to drive the plunger of each syringe at acontrolled rate to force the PCR mixture from the syringe into therespective tee. A tee enables a respective syringe to create a pluralityof microreactors (each preferably containing a single bead on averageand an effective single nucleic acid template) in the emulsion oil.Thereafter, the emulsion flow (with microreactors) is sent to a thermalprocessor so that the nucleic acid template supplied in eachmicroreactor may be amplified for nucleic acid amplification. FIG. 3illustrates an example of beads (see arrows) suspended in individualmicroreactors.

FIG. 4 illustrates a general schematic of an injection tee emulsifier400 that may be used with some of the embodiments of the presentinvention, which allow, for example, droplet generation rates on theorder of 500 to 1000 per second or more, and FIGS. 5A-5F illustratevarious views of particular injection tee emulsifiers according to someembodiments of the invention.

As shown in FIG. 4, the emulsifier may include a first inlet 402 of afirst conduit 404 for receiving an emulsion oil, a narrowed diameterarea 406 provided along the first conduit and an outlet 408 of the firstconduit. Preferably, the first conduit is provided in a horizontalposition thereby establishing a cross-flow of emulsion oil through thenarrowed area. Moreover, a diameter of the narrowed area is preferablybetween 100 μm and 600 μm, more preferably between 200 μm and 400 μm,and most preferably approximately 300 μm.

The emulsifier 400 also includes a second inlet 410 of a second conduit412 for directing amplification reaction/bead mixture into theapparatus. A tubular orifice 414 is provided at a terminus of the secondconduit, and is open to the narrowed area 406 of the first conduit. Theorifice preferably includes a diameter of between about 10 μm to about200 μm, and more preferably between 75 μm and 150 μm, and mostpreferably about 120 μm. Preferably, the second inlet, conduit andorifice are provided in a vertical arrangement relative to the preferredhorizontal arrangement of the first conduit (i.e., the first conduit andsecond conduit/orifice may be orthagonal to one another), although anyorientation can work. The orifice enables a plurality of amplificationreaction mixture droplets (i.e., microreactors), to be created as thereaction mixture enters the oil flow. A plurality of such microreactorseach preferably include on average a single bead and an effective singlenucleic acid template.

With reference to FIG. 5D, a particular injection tee includes a syringeexit area 502 d, an aqueous phase (PCR solution) inlet area 504 d, atapered area of injection port 506 d, a straight area of injection port508 d, an emulsion exit 510 d, an emulsion oil inlet 512 d, a taperedoil acceleration area 514 d (i.e., nozzle), a constant speed, highvelocity (narrowed) area 516 d, a partial diffusion area 518 d and adiffuser step 520 d.

Accordingly, when the emulsion oil enters a respective injection tee, itenters a progressively narrower region, and is thus accelerated to ahigher velocity (e.g., 30 times its initial velocity). The PCR/beadsolution (i.e., an aqueous phase material) is then injected atpreferably a constant and controlled rate, preferably between about 5μl-100 μl per minute, and preferably about 20 μl/minute. One of skill inthe art will appreciate that other relative flowrates for the twocomponent fluid sources may be used in accordance, for example, with thespecifications of a thermal processing device for PCR amplification.

The shearing force of the high velocity oil breaks off the PCR/beadstream into individual droplets as it is being injected, each preferablyincluding a single bead. As the newly formed droplets move downstream,the velocity of the flow may be gradually reduced in a diffuser area(see partial diffuser area 522, FIG. 5D). After the partial diffuser,the flow encounters an abrupt step (520, FIG. 5D), which causes thedroplets to break away from the wall and enter the central area of theflow stream. The flow then exits the injection tee with the dropletsevenly distributed throughout the flow.

The examples of injection tees illustrated in FIGS. 5A-5D may be made tofit a disposable syringe, and may be manufactured via plastic injectionmolding. In some embodiments of the invention, the selection of anappropriate plastic material is critical to impart the desired functionof the mixing tee. Specifically, the surface of the material mustpreferentially wet with oil rather than water. If this is not the case,the incoming stream of aqueous material will flow along the inside wallof the high-velocity area of the tee in a continuous stream, rather thanbe sheared into the desired sized droplets. Polypropylene, for example,is a plastic material that has meets these requirements. An additionalrequirement for the tee is that the internal geometry must cause thenewly formed emulsion droplets to leave the wall of the oil conduit(where they are formed) and migrate to the central area of the flowstream. If this does not occur, the droplets will be too close togetherand the risk of collisions and coalescence will be high. To that end,present examples of the injection tees include internal geometryfeatures which induce the emulsion droplets to separate from walls andflow into the central area of the conduit.

The PCR/bead mixture is provided to the injection tee using a syringepump, an example of which is illustrated in FIGS. 6A-6E. As illustratedgenerally in FIGS. 6A-6C, the syringe pump 600 generally includes one ormore syringes 602, a syringe holder block 604, guide rods 606, a drivescrew 608, a motor 610, a base 612, a slider assembly 614 and a pivotingdoor assembly (which may be spring-loaded) 616. The slider assembly isdriven by the drive screw, which in turn simultaneously drives eachplunger of each syringe into the syringe body to drive out the contentsof the syringe. The motor, which may be a stepper motor, turns a drivepulley 618, which drives a main drive pulley 620 via belt 622, whichrotates the drive screw to move the slider assembly (to move the syringeplungers).

To insure the beads are evenly distributed throughout the solutionwithin each syringe a mixing mechanism may also be included with thesyringe pump. As shown in FIG. 6D, a rotating element 624 (drum) may beincluded (in this case, positioned in the pivoting door assembly) whichincludes a helical line of magnets 626 along a portion of the drumsurface positioned adjacent each syringe body. In an operating position,the rotating element rotates so that the helical line of magnets comeinto close proximity to the bodies of the syringes as they pass by. Anelectric motor (not shown) with a gear reduction unit may be used topower the rotating element and may be mounted inside the rotatingelement (being secured to the door frame). The plurality of magnets mayalso be represented by a single, helically wound magnet-strip (or otherformed magnet, which is helically arranged around the rotating element),but a plurality of individual magnets is preferred.

The magnets 626 in the rotating element are preferably oriented so thatthe fields are directed out radially from the rotating element.Preferably, a majority (and most preferably, all) of the magnets havethe same polarity orientation.

As the helically mounted line of magnets 626 pass by the body of eachsyringe, a magnetic ball 632 a, 632 b, 632 c, included inside eachsyringe body is moved from its lowest position adjacent the plunger 634toward a higher position, which may be adjacent the nozzle area 636,successively, higher and higher by each magnet. The ball is releasedonce the highest magnet in the helical series moves away from thesyringe body and then drops to the bottom (i.e., adjacent the plunger)of the syringe. This motion of the mixing ball will occur regardless ofwhere the syringe plunger is located. The frequency and velocity of themixing ball may be controlled by the rotational speed of the rotatingelement. Preferably, as the syringe becomes less full, the ball movesmore quickly to accomplish the same mixing effect. More than one helicalpattern of magnets may be used to allow different mixing rates anddisplacements as the plunger moves through different areas of thesyringe. For example, it is desirable that the mixing action does notdisturb the injection tee, so one series of magnets may start at thelowest point of the plunger and only go part way up the syringe body,while a second line of magnets may start at the middle and continue tothe top. The lower magnets would work when the plunger is low, and theupper portion of the magnets would only work when the plunger is in theupper range of its motion. Another variation is for the helix of magnetsto go both upward and downward, thereby controlling the motionthroughout and allowing the mechanism to work in any orientation.

To insure the beads do not clog the nozzle in the mixing tee, someembodiments of the present invention make use of an anti-cloggingdevice. More particularly, some embodiments of the present include asonic (vibratory), anti-clogging mechanism (FIG. 6E) for reliablyfeeding solid particles at high concentrations through the nozzle of themixing tee. As shown in FIGS. 6E, each plunger 632 a, 632 b, 632 c maybe fitted with a piezo-electric actuator 638 (i.e., sonic actuator),which is provided in the slider assembly of the syringe pump. Thepiezo-electric actuator(s) are driven electronically at a desiredfrequency (between about 50 Hz and 1000 Hz, and preferably about 300 Hz)and displacement (between about 1μ-100μ, and preferably about 15μ) toeffectively keep the particles in the nozzle in constant motion toprevent clumping & clogging of the beads in the nozzle area.Electromagnetic actuators can also be used to create & impart the energyto the syringes.

Without the use of the sonic actuators according to the presentinvention, large amounts of viscosity enhancers and surfactants would berequired to prevent particles from clogging the nozzles, and such asystem would still only be marginally reliable. Another importantfunction of the vibratory mechanical excitation of the syringe plungersis that it prevents the rubber syringe plungers from sticking to theinternal body of the syringe. If this happens, a slip-stick intermittentmotion of the syringe plunger will occur, causing the bead/PCR solutionto be fed at a variable rate, causing droplet size control to beimpossible.

The flow exiting from the emulsifier, via a conduit (for example) may bethen run through a thermal processing device, which exposes thecontinuous flow to alternate zones of a higher temperature and lowertemperature (e.g., a heating zone and a cooling zone) for PCRamplification. Examples of such thermal processing devices areillustrated in FIGS. 7A-7B and FIGS. 8A-8C. Some embodiments of thethermal processors according to the present invention allow for rapid,simultaneous and separate PCR amplification of millions of DNAfragments, resulting in clonal solid phase products. Over 12 million(for example) separate DNA fragments can be separately amplified in onebatch.

For example, a stainless steel conduit, including an inlet 702 and anoutlet 704, containing the emulsified flow (e.g., 400 as shown in FIG.4) may be helically wound around a mandrel type thermal processingdevice 700 (FIG. 7A-7B). One side or portion of the mandrel comprises afirst temperature zone 706 which may include a heater or heat transferelement 708, surrounded by an insulation material 709 (for example) anda second side or portion of the mandrel comprises a second temperaturezone 710 which includes a temperature lower than the temperature of thefirst temperature zone. The second temperature zone may include acooling element (e.g., water jacket, air circulation fan, and the like)to cool the continuous flow, but may also include a resistance heater711 (for example) to maintain a certain predetermined temperature. Apreheating zone 712 may also be included, prior to the flow reaching thefirst temperature zone.

Alternatively, the second temperature zone may also include a heatingelement, since the zone typically, for PCR amplification, maintains atemperature of between approximately 60-70 degrees C., which isconsiderable higher than room temperature of 23 degrees C. Such anexample is shown in the thermal processor illustrated in FIGS. 8A-8C. Ifa heat transfer element, such as a thermoelectric device, is used toprovide heat to the first temperature zone, the heat will come from thesecond zone, which must have a heating element to maintain temperature.This is the desirable approach because it generates the least possibleamount of waste heat and therefore has the lowest energy consumption andthe lowest cooling requirement.

In this example, the thermal processing device 800 allows alternatingsections of a conduit 802 to be positioned adjacent opposed (forexample) linear first temperature 804 and second temperature 806 zones.One or more thermoelectric heat transfer elements 808 of the thermalprocessing device may provide heat to the continuous flow along portionsof the conduit. Resistance heaters 810 may be positioned adjacent thesecond temperature zone 806, so that the conduit sections positionedadjacent the second temperature zone is maintained at, for example,between a temperature of, for example, 60-70 degrees C. The secondtemperature may maintained by both adding heat from the heating elementswhen required, and also by removing heat in the second zone, using (forexample) a fan to move air across the second zone. Another feature thatmay be included is an exposed area of conduit between the first andsecond temperature zone, which allows the conduit to be directly cooledby the fan, rather than via the second temperature zone block.

In this example, the resistance heaters may also provide thermal energyfor the first temperature zone. Blocks 812 may be used as thermalconductors to conduct heat to and from the various zones and to and fromthe conduit, and may be fabricated from any material useful for heating,e.g., metal such as aluminum, copper, and the like. The blocks may bedesigned to be a precise fit around the conduit so that thermal energymay be efficiently transferred between the blocks and the conduit. Athermal grease may be added between these two elements to furtherimprove the thermal conductivity of the interface. Insulation 814 mayalso be used to help maintain temperature of the high temperature zone,or any other area of the thermal processing device. In one example, theconduit may extend 46 cm for each amplification cycle, for a total of 35cycles. In another example, the conduit is 67 cm for each cycle, and atotal of 60 cycles are used. The conduit is made in groups of fivecycles, so that sufficient fasteners may be used to ensure that theblocks are tightly clamped around the conduit. To allow a pre-heatingstep, an additional length of conduit may be added in the beginning thatis exposed only to the high temperature zone. This may be included forthe purpose of activating an enzyme required for PCR amplification.Further still, a distal end of the conduit may be adapted to allow forsample collection, e.g., into a bead filtering device or a collectioncontainer.

For PCR, the temperature in the first temperature zone of between 90 and100 degrees C. may be used to melt duplex nucleic acid (e.g., 94° C.),while the 60-70 degrees C. temperature of the second temperature zone ischosen for primer annealing and extension (e.g., 65° C.), for example.

While the delivery of the emulsion components can be accomplished by anymanual or automatic delivery means, preferably a pump system is used. Asillustrative examples, delivery can be obtained by various pumps,including syringe pumps and mechanical pumps, e.g., HPLC pumps (see,e.g., Gilson, Inc., Middleton, Wis.; ESA, Inc., Chelmsford, Mass.; JascoInc, Easton, Md.). The preferred means is a rotary annular gear pump.

Exemplary heating devices for the apparatus include, but are not limitedto, cartridge heaters (see, e.g., Omega Engineering, Inc., Stanford,Conn.; Delta-t Max, Greenland N.H.), resistive heaters (see, e.g., MincoProducts, Inc., Minneapolis, Minn.), and thermoelectric heaters,including Peltier devices (see, e.g., Ferrotec, Nashua N.H.). In variousaspects, the heating devices for the apparatus can be embedded in theheating blocks or mounted on the surface of the blocks. Temperaturemonitors may also be used with the apparatus, including real-timeproportional temperature controllers, PID (proportional, integral, andderivative) digital controllers, in combination with temperature sensingelements such as thermocouples, thermistors, or any other suitabledevice (see, e.g., Watlow Electric Mfg. Co., St. Louis, Mo.).

The conduit material may be fabricated out of any compatible tubingmaterial for amplification (in particular, thermal amplification), suchas stainless steel, Polytetrafluoroethylene (PTFE; e.g., Teflon), andfused silica. Preferably, stainless steel tubing is used for its thermalconductivity and corrosion resistance.

It will be understood that other means for controlling amplification forthe apparatus are also possible. For example, fluids can be circulatedfrom constant temperature reservoirs, in particular, hot oil baths (see,e.g., Nakano et al., 1994, Biosci. Biotech. Biochem. 58:349-352), andhot water baths (see, e.g., Curcio and Roaeraade, 2003, Anal. Chem.75:1-7). In addition, it is possible to perform continuous flowamplification on the surface of a chip (see, e.g., Kopp et al., 1998,Science 280:1046-1049; Schneegas et al., 2001, Lab on a Chip 1:42-49).For example, a silicon or glass chip can be modified to include thinfilm transducers to heat different sections of the chip to differenttemperatures. Alternatively, a chip can be placed across a row ofheating blocks, where each block is heated to a different temperature.The heated sections of the chips can allow for denaturation (e.g., 95°C.), primer annealing (e.g., 58° C. or 60° C.), and primer extension(e.g., 72° C. or 77° C.) steps in the amplification reaction. Inaddition, fluid channels may be added to the chip (e.g., by etching,molding, imprinting, or adhesives) to allow for buffer and sample input,temperature cycling, and product output. The buffers and samples can bedelivered, for example, by precision syringe pumps, and theamplification products can be collected into microfuge tubes,microwells, or other reservoirs. These methods, however, do not providefor the separate amplification of large numbers (thousands or millions)of different DNA fragment templates, which is the critical advantage ofthis invention.

Bead Recovery

Following amplification of the nucleic acid template and the attachmentof amplification copies to the bead, the beads must be recovered. If afilter element is at the exit of the first conduit, the filter may beremoved from the system, and beads may be back-flushed out of the filterusing reverse flow. The beads may alternatively be washed & processedwhile they are still in the filter, by attaching the filter to a syringewith the bead side exposed to the syringe chamber, and pulling andpushing various wash reagents through the filter and in and out of thesyringe.

Alternatively, the emulsion exiting the flow system may be collected ina vessel, and subsequently spun in a centrifuge, which will leave thebeads at the bottom provided they are denser than the oil. The oil maythen be removed from above the beads, and the beads may be recoveredfrom the bottom of the vessel. This procedure may also be used withoutthe centrifuge, if sufficient time is allowed for the beads to settle bygravity.

Purifying the Beads

After PCR amplification, the beads may be isolated from themicroreactors and used for sequencing. The sequencing steps arepreferably performed on each individual bead. However, this method,while commercially viable and technically feasible, may not be mosteffective because a portion of the beads will be “negative” beads (i.e.,beads without amplified nucleic acid attached). This is because the DNAtemplate material is delivered to the PCR solution or the beads bydilution, and it is inevitable that at least some of the beads do notget a starting copy for amplification. In such cases, an optionalprocess outlined below may be used to remove negative beads prior todistribution onto multiwell (e.g., picotiter) plates.

EXAMPLES Binding Nucleic Acid Template to Capture Beads

This example describes preparation of a population of beads thatpreferably have only one unique nucleic acid template attached thereto.Successful clonal amplification depends on the delivery of a controllednumber of template species to each bead. Delivery of excess species canresult in PCR amplification of a mixed template population, preventinggeneration of meaningful sequence data while a deficiency of specieswill result in fewer wells containing template for sequencing. This canreduce the extent of genome coverage provided by the sequencing phase.As a result, it is preferred that the template concentration beaccurately determined through replicated quantitation.

Template Quality Control

The success of the Emulsion PCR reaction is related to the quality ofthe template species. Regardless of the care and detail paid to theamplification phase, poor quality templates will impede successfulamplification and the generation of meaningful sequence data. To preventunnecessary loss of time and money, it is important to check the qualityof the template material before initiating the PCR phase of the process.Preferably, the template library should pass two quality control stepsbefore it is used in Emulsion PCR. Its concentration and thedistribution of products it contains should be determined. Ideally, thelibrary should appear as a heterogeneous population of fragments withlittle or no visible adapter dimers (e.g., ˜90 bases). Also,amplification with PCR primers should result in a product smear ranging,for example, from 300 to 500 bp. Absence of amplification product mayreflect failure to properly ligate the adaptors to the template, whilethe presence of a single band of any size may reflect contamination ofthe template.

Continuous Flow PCR Amplification

A linear emulsifier included an internal diameter of 300 μm for an oilpassage and an internal diameter of 120 μm for the bead/PCR solutionoutlet (see FIG. 4). The emulsion oil flow rate was set at 2 ml/min,while the PCR solution flow rate was set at 5 μl/min. The droplet(microreactor) size range was 80 μm to 120 μm (270 pl to 900 pl).Droplets were generated at a rate of 55/sec to 180/sec. Bead size was 25μm to 30 μm, while bead density was 1 bead/nl. The flow tube internaldiameter was 2.4 mm. The length of tube for one cycle was 46 cm. EachPCR cycle was timed at 64 sec, which included 35 cycles plus a pre-heatstep taking 2 min. The total time for the PCR reaction was 39 minutes.

Solution Phase Reaction Mix:

-   1×Hi FI Buffer-   1 mM dNTPs-   2.5 mM MgSO4-   1 uM forward Primer (MMP 1a)-   1 uM Reverse Primer (MMP1b)-   0.01% tween-80-   0.1% BSA-   0.15U/μl Hi Fi Taq

Added 3 and 30 copies of TF7 per nl PCR mix

For Bead experiment used same reaction mix only added 0.333% 4M weightPEO and added 3,600 copies/nl of TF7 into the solution. Added beads at 1bead/nl.

Copy per nl Amplification Factor   3  53M  30 7.6M 3600(+beads) 9,600

Pyrosequencing of beads from reaction showed clear TF7 sequence.

The total amplification obtained was 50,000,000×, corresponding to 1.66×amplification per PCR cycle.

Another Example of Emulsion Flow PCR

The PCR amplification mixture used contained 1×High Fidelity Buffer (60mM Tris-SO4 pH 8.9, 18 mM Ammonium Sulfate, (Invitrogen)), 1 mM dNTPs(Pierce), 0.625 mM forward primer, 0.078 mM reverse primer (IDT), 0.25%agrimer AL10-LC (ISP Technologies), 5% PEG-8000 (Acros), 0.02% BSA(Sigma), 0.003 U/ul inorganic pyrophosphotase (NEB), 0.15 U/ul PlatinumHigh Fidelity Taq (Invitrogen).

The library of interest, E. coli, was added in three replicates to 1.8million capture beads in a minimal volume and resuspended by vortexing.This mixture was then added to 900 μl of the PCR mixture. This solutionwas then loaded into a 1 ml syringe that contained a 4.1 mm plasticcoated magnetic mixing ball. Three identical syringes were then loadedin series onto the “Flow PCR Unit”.

The emulsion flow PCR system included an internal diameter of 300 μm foran oil passage and an internal diameter of 120 μm for the bead/PCRsolution outlet (see FIG. 4). The emulsion oil flow rate was set at 2.4ml/min, while the PCR solution flow rate was set at 15 μl/min persyringe. Three syringes were used. The droplet (microreactor) size rangewas 80 μm to 120 μm (270 pl to 900 pl). Droplets were generated at arate of 280/sec to 920/sec per syringe. Bead size was 25 μm to 30 μm,while bead density was 2 beads/nl. The flow tube internal diameter was2.4 mm. The length of tube for one cycle was 67 cm. Each PCR cycle wastimed at 60 sec, and there were 60 cycles plus a pre-heat step taking 2min. The total time for the PCR reaction was 62 minutes. A 15 μm mesh,25 mm diameter filter was used to capture the beads as they exited thethermal processor.

2.42 million beads were recovered from the filter and then enriched.

Enrichment Protocol Summary:

An enrichment primer (containing both the amplification and sequencingprimer regions) is annealed to the beads. The beads are then washed inbuffer containing 2M NaCl and Tris pH 7.5 and then mixed with 1 micronbiotinylated Seramag beads. This mixture is incubated at RT for threeminutes on a rotator and then pelleted at 2,000 rpm in amicrocentrifuge. The beads are resuspended by hand vortexing and thenincubated on ice for 5 minutes. The mixture is washed on a DYNAL-MPCmagnet to remove unannealed material and then NaOH is added to removethe annealed oligo. “Enriched” beads are then recovered by washing in1×annealing buffer. 2.07 million beads were recovered from theenrichment process.

700,000 of the enriched beads were then sequenced. 414,557 of thesebeads had recognizable DNA sequences on them, and 206,000 of those beadshad a sequence that averaged 90.5 base pairs long and were mapped to alocation on the E. coli genome.

Throughout this specification, various patents, published patentapplications and scientific references are cited to describe the stateand content of the art. Those disclosures, in their entireties, arehereby incorporated into the present specification by reference.

1.-9. (canceled)
 10. An apparatus for amplifying genetic materialcomprising: at least one fluid delivery device; at least one firsttemperature zone to cycle a plurality of aqueous microreactors formedfrom a water-in-oil emulsion to a first temperature; at least one secondtemperature zone to cycle the plurality of aqueous microreactors to asecond temperature lower than the first temperature; a first conduit forflowing at least a continuous stream of oil therein from a firstreservoir; and a second conduit for flowing at least a water based PCRamplification reaction mixture including one or more species of nucleicacid templates, a plurality of beads, and PCR reagents necessary foramplification of the one or more species of nucleic acid templates froma second reservoir out of an orifice and into the first conduit, therebycreating the water-in-oil emulsion in the continuous stream of oil,wherein the plurality of aqueous microreactors each include the one ormore species of nucleic acid templates, a single bead capable ofcapturing the one or more nucleic acid templates, and sufficient PCRreagents to amplify the copy number of the one or more species ofnucleic acid templates.
 11. The apparatus for amplifying geneticmaterial according to claim 10, wherein downstream of the orifice, alength of the first conduit from a starting position to an endingposition is arranged relative to at least the first and secondtemperature zones such that the length of the first conduit is expose toalternating processes of heating and cooling at a temperature and timesufficient to amplify the one or more species of nucleic acid templatesby polymerase chain reaction.
 12. The apparatus according to claim 10,wherein the orifice is sized between 50 μm to 300 μm.
 13. The apparatusaccording to claim 10, wherein the orifice is sized to approximately 150μm.
 14. The apparatus according to claim 10, wherein an intersectionarea of the orifice and the first conduit includes a diameter of betweengreater than 50 μm to about 800 μm.
 15. The apparatus according to claim10, wherein an intersection area of the orifice and the first conduitincludes a diameter of about 400 μm.
 16. The apparatus according toclaim 10, further comprising collecting means for collecting amplifiedcopies of the one or more species of nucleic acid templates from thefirst conduit downstream of the heating and cooling sources.
 17. Theapparatus according to claim 16, wherein the collecting means comprisesa filter.
 18. The apparatus according to claim 10, wherein the first andsecond temperature zones are circumferentially arranged on opposed sidesof a curved surface, wherein the length of the first conduit ishelically wound around the curved surface to provide alternatingportions of the length of first conduit adjacent either the firsttemperature zone or the second temperature zone.
 19. The apparatusaccording to claim 18, wherein the curved surface comprises a mandrel.20. The apparatus according to claim 10, wherein the temperature zonesare arranged along opposed heating and cooling linear surfaces,respectively, wherein the length of first conduit is wound along theopposed surfaces such that portions of the length of first conduit arealternately exposed to the heating and cooling surfaces a plurality oftimes.
 21. The apparatus according to claim 20, where the linearsurfaces are substantially vertical or horizontal.
 22. The apparatusaccording to claim 10, wherein the aqueous microreactors have an averagesize of between approximately 50 to approximately 250 μm in diameter.23. The apparatus according to claim 10, wherein the one or more speciesof nucleic acid templates are selected from the group consisting ofgenomic DNA, cDNA, episomal DNA, BAC DNA, and YAC DNA.
 24. The apparatusaccording to claim 10, wherein each bead has a diameter of betweenapproximately 2 μm to approximately 100 μm.
 25. The apparatus accordingto claim 24, wherein the bead is selected from the group consisting of asepharose bead, a solid bead and a monodisperse bead.
 26. A cross-flowemulsification apparatus comprising: a first inlet for receiving an oilflow from a first conduit; an outlet for directing a water-in-oilemulsion out of the apparatus; a second inlet for receiving a waterbased PCR amplification reaction mixture comprising one or more speciesof nucleic acid templates, a plurality of beads and PCR reagentsnecessary for amplification of the one or more species of nucleic acidtemplate; and an orifice for delivering the PCR amplification reactionmixture from the second conduit into the first conduit, to form aplurality of water-in-oil droplets comprising aqueous microreactors,wherein the aqueous microreactors each include the one or more speciesof nucleic acid templates, a single bead capable of capturing the one ormore nucleic acid templates, and sufficient PCR reagents to amplify thecopy number of the one or more species of nucleic acid templates. 27.The apparatus according to claim 26, wherein the orifice delivers theplurality of water-in-oil droplets into the first conduit at a narrowedregion provided in the first conduit.
 28. The apparatus according toclaim 27, wherein the narrowed region includes a diameter between about40 μm and 600 μm.
 29. The apparatus according to claim 27, wherein thenarrowed region includes a diameter of approximately 300 μm.
 30. Theapparatus according to claim 26, wherein the orifice includes a diameterbetween approximately 60 μm and about 300 μm.
 31. The apparatusaccording to claim 26, wherein the orifice includes a diameter ofapproximately 120 μm.
 32. The apparatus according to claim 26, whereinthe aqueous microreactors have an average size of between approximately50 to approximately 250 μm in diameter.
 33. The apparatus according toclaim 26, wherein the one or more species of nucleic acid templates areselected from the group consisting of genomic DNA, cDNA, episomal DNA,BAC DNA, and YAC DNA.
 34. The apparatus according to claim 26, whereineach bead has a diameter of between approximately 2 μm to approximately100 μm.
 35. The apparatus according to claim 34, wherein each bead is asepharose bead.
 36. (canceled)
 37. An emulsion generator comprising: anemulsion oil supply; at least one syringe including a body and a plungerfor dispensing a mixture for emulsifying into the emulsion oil; across-flow emulsification device for emulsifying the mixture, the deviceincluding an input attached to the output of the syringe; and a syringepump including an actuator capable of oscillating the plunger of the atleast one syringe micrometer distances at a predetermined frequencyalong a length of travel of the plunger within the syringe body of theat least one syringe.
 38. The emulsion generator according to claim 37,wherein the actuator is capable of moving the plunger between about 5 μmand about 50 μm.
 39. The emulsion generator according to claim 37,wherein the desired frequency is between about 1 Hz and 500 Hz.
 40. Amethod for substantially reducing clogging of a nozzle in a syringepump, comprising: providing a syringe pump having at least one syringeincluding a body for dispensing a mixture of micron or less sizedparticles suspended in a medium, an actuator, a plunger having a plungeraxis and an exit nozzle; and oscillating the plunger of the syringealong the axis of the plunger for micrometer distances at apredetermined frequency along a length of travel of the plunger withinthe syringe body.
 41. The method according to claim 40, wherein theactuator is capable of moving the plunger between about 5 μm and about50 μm.
 42. The method according to claim 40, wherein the predeterminedfrequency is between about 1 Hz and 500 Hz.
 43. The emulsion generatoraccording to claim 37, further comprising: a magnetically-attractivemixing element disposed in the body of the syringe; and a device capableof moving an external magnetic force axially along body of the syringewhile in close proximity to the syringe body.
 44. The emulsion generatoraccording to claim 43, further comprising: a rotating drum having amagnet helically wound along the surface of the drum, wherein thesurface of the drum is positioned adjacent the body of the syringe. 45.The emulsion generator according to claim 44, wherein the magnetcomprises a plurality of individual magnets helically spaced along thesurface of the drum.
 46. The emulsion generator according to claim 43,wherein the mixing element comprises a plastic coated metallic ball. 47.A syringe pump comprising: an area for receiving at least one syringe,wherein the syringe includes a body and a plunger having a plunger axis,the syringe for dispensing a mixture for emulsification into an emulsionoil; and an actuator capable of oscillating the plunger of the at leastone syringe along the plunger axis micrometer distances at apredetermined frequency along a length of travel of the plunger withinbody of the at least one syringe.
 48. The syringe pump according toclaim 47, wherein the actuator is capable of moving the plunger betweenabout 5 μm and about 50 μm.
 49. The syringe pump according to claim 47,wherein the predetermined frequency is between about 1 Hz and 500 Hz.50. The syringe pump according to claim 47, further comprising: amagnetically attractive mixing element disposed in the body of thesyringe; and a rotating drum having a magnet helically wound along thesurface of the drum, wherein the surface of the drum is positionedadjacent to the body of the syringe.
 51. The syringe pump according toclaim 50, wherein the magnet comprises a plurality of individual magnetshelically spaced along the surface of the drum.
 52. The syringe pumpaccording to claim 50, wherein the mixing element comprises a plasticcoated metallic ball.